Oxidative stress and apoptosis are considered common mediators of disease, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington disease (HD), Amyotrophic Lateral Sclerosis (ALS), ischemia and stroke, spinal cord trauma and head injury, cardiovascular diseases and inflammatory diseases. Despite the growing amount of information regarding the pathophysiological role of oxidative insult in the apoptotic cell death process, little effort has been made to identify the key cell signaling molecule that contributes to the cell death.
Parkinson's disease (PD) is a major common neurodegenerative disorder affecting more than 1% of the population over the age of 60 in the US (Allam et al., 2005; West et al., 2005). Selective degeneration of nigral dopaminergic neurons is the primary pathology of PD (Przedborski, 2005). Both clinical and experimental evidence clearly demonstrates that oxidative stress and apoptosis are key cellular mechanisms that contribute to the selective nigral neuronal loss (Maguire-Zeiss et al., 2005). However, the key cellular target that mediates the nigral apoptotic cell death process following oxidative insult is not completely understood.
Recently, it was reported by the inventors that proteolytic activation of PKCδ, a member of the novel PKC isoform family, plays a key role in apoptotic cell death of dopaminergic neurons in a cell culture model of PD as well as oxidative stress models (Anantharam et al., 2002; Kaul et al., 2003; Kitazawa et al., 2003; Yang et al., 2004; Latchoumycandane et al., 2005). It was demonstrated that blockade of PKCδ activation by the kinase dominant negative mutant, cleavage-resistant mutant or siRNA almost completely prevented the nigral cell death (Kaul et al., 2003; Kitazawa et al., 2003; Anantharam et al., 2004; Yang et al., 2004; Latchoumycandane et al., 2005). PKCδ is proteolytically cleaved by caspase-3 at the 324DIPD327 (SEQ ID NO:5) residue, resulting in 41-kDa catalytic and 38-kDa regulatory subunits, leading to a persistent activation of the kinase (Kaul et al., 2003; Anantharam et al., 2004; Yang et al., 2004).
Protein kinase C (PKC) belongs to a family of serine threonine protein kinases. To date, twelve isoforms in the PKC subfamily have been identified. Kanthasamy et al., 2003; Antioxidants & Redox Signaling, 5: 609-620. One such isoform is protein kinase C delta (PKCδ). Martelli A M, Mazzotti G, Capitani S, Nuclear protein kinase C isoforms and apoptosis. Eur J Histochem. 2004; 48(1):89-94.
PKCδ was originally discovered by Gschwendt et al. in 1986, Gschwendt M, Kittstein W, and Marks F. A novel type of phorbol ester-dependent protein phosphorylation in the particulate fraction of mouse epidermis. Biochem Biophys Res Commun. 137: 766-74, 1986, and cloned from a rat brain cDNA library the following year. Kurkinen K M, Keinanen R A, Karhu R, and Koistinaho J. Genomic structure and chromosomal localization of the rat protein kinase Cdelta-gene. Gene 242: 115-23, 2000, Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, and Nishizuka Y. Identification of three additional members of rat protein kinase C family: delta-, epsilon- and zeta-subspecies. FEBS Lett. 226: 125-8, 1987. The PKCδ gene is localized on human chromosome 3, Huppi K, Siwarski D, Goodnight J, and Mischak H. Assignment of the protein kinase C delta polypeptide gene (PRKCD) to human chromosome 3 and mouse chromosome 14. Genomics 19: 161-2, 1994, rat chromosome 16, Kurkinen K M, Keinanen R A, Karhu R, and Koistinaho J. Genomic structure and chromosomal localization of the rat protein kinase Cdelta-gene. Gene 242: 115-23, 2000, and mouse chromosome 14, Huppi K, Siwarski D, Goodnight J, and Mischak H. Assignment of the protein kinase C delta polypeptide gene (PRKCD) to human chromosome 3 and mouse chromosome 14. Genomics. 19: 161-2, 1994.
There exists a substantial body of evidence that indicates that PKCδ plays a fundamental role in apoptosis. PKCδ has been shown to accumulate in the nucleus of C5 cells, in response to etoposide treatment. Martelli A M, Mazzotti G, Capitani S, Nuclear protein kinase C isoforms and apoptosis. Eur J Histochem. 2004; 48(1):89-94. Overexpression of PKCδ-catalytic fragment results in nuclear localization of the PKCδ fragment and apoptosis. Martelli A M, Mazzotti G, Capitani S, Nuclear protein kinase C isoforms and apoptosis. Eur J Histochem. 2004; 48(1):89-94. It is believed that active PKCδ binds to the carboxyl-terminus of DNA-dependent protein kinase (DNA-PK), an enzyme involved in the repair of DNA strand breaks. Once bound, the active PKCδ binds to and phosphorylates the DNA-PK. Consequently, the DNA-PK dissociates from the DNA, impedes the repair of DNA strand breaks, and results in DNA fragmentation, one of the hallmarks of apoptosis.
Apoptosis, a genetically programmed form of cell death, is required for normal development, tissue homeostasis and the elimination of damaged cells. However, an increase or decrease in apoptosis may contribute to the pathology of a wide range of disorders and diseases. It has been proposed that apoptosis plays a central role in several human neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington disease (HD), amyotrophic lateral sclerosis (ALS), ischemia and stroke, cardiovascular diseases, inflammatory diseases, spinal cord trauma, and head injury.
It is an object of the present invention to provide novel compositions and methods which inhibit the caspase mediated cleavage of PKCδ.
It is another object of the invention to provide a novel target site for therapeutic intervention to prevent neurodegenerative apoptosis.
It is yet another object of the invention to provide pharmaceutical composition for the treatment, or amelioration of symptoms or protection from further damage in Alzheimer's disease (AD), Parkinson's disease (PD), Huntington disease (HD), Amyotrophic Lateral Sclerosis (ALS), ischemia and stroke, spinal cord trauma and head injury, cardiovascular diseases, inflammatory diseases, and other disease states associated with oxidative stress and apoptosis.
It is yet another object of the invention to prevent oxidative stress and apoptosis in an animal.